Macs cd14 isolation kit

MoDCs were differentiated from CD14⁺ monocytes isolated from PBMCs. CD14⁺ monocytes were isolated using the MACS CD14⁺ isolation kit (130-050-201, Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s protocol. Per 1 × 10⁷ cells, 80 µL of wash buffer and 20 µL of CD14⁺ beads were added to the cells. After 15 min of incubation at 4 °C, the PBMCs were washed with 50 mL of washing buffer (5 min, 4 °C, 1500 rpm). The pellet was resuspended in 0.5 mL per 1 × 10⁶ cells. Then, 0.5 mL of the cell suspension was added to LS columns (130-042-901, Miltenyi Biotec), which were pre-washed with washing buffer (2 mL). After the flowthrough was collected the columns were washed three times with 1 mL of washing buffer. The columns were taken off the magnet and the CD14⁺ cells were flushed out with 2 mL of washing buffer and counted. The CD14⁺ cells were resuspended in 0.5 million cells/mL in X-VIVO medium (Lonza, Bazel, Zwitserland) + 2% human serum (Sanquin), IL-4 (300 IU/mL, 130-093-924, Miltenyi Biotec) and GM-CSF (450 U/µL, 130-093-868, Miltenyi Biotec). To obtain immature moDCs, CD14⁺ cells were cultured for six days in 96-well plates (100,000 cells in 200 µL per well). After three days, cytokines IL-4 (300 IU/mL) and GM-CSF (450 U/µL) were again added.

Monocyte-derived DCs were obtained from a healthy human blood donor following a standard protocol [26 (link)]. Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats in lymphocyte separation medium (Corning) by density gradient centrifugation at 1500 rpm. Then, CD14+ monocytes were purified by using a MACS CD14 isolation kit (Miltenyi Biotech). Monocytes were then differentiated into DCs by incubating the cells during 5 days, at 37°C, in growth media containing RPMI 1640 (Invitrogen/Gibco), 8% FBS (Hyclone), 2 mM glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 500 U/mL hGM-CSF (PeproTech), and 1000 U/mL hIL-4 (PeproTech). Before infection, DCs were pretreated for 1 h at 37°C with RPMI medium containing the NFAT inhibitors cyclosporine A (CsA, Sigma, 1 μM) [27 (link)] or the VIVIT peptide (Tocris Biosciences, 100 μM) [28 (link)]. Then, the cells were infected with the NC virus grown in embryonated chicken eggs as described previously [29 (link)], diluted in DMEM, and added directly to pelleted cells at a MOI of 1. After incubation of 40 minutes at RT, fresh RPMI medium containing CsA (1 μM) or the VIVIT peptide (100 μM) was added back, and the cells were incubated at 37°C during 2 h. Mock-infected cells underwent the same experimental procedure.

Healthy donors enrolled in the study gave written informed consent prior to the procedure. The study protocol was approved by the Institutional Review Board of Severance Hospital and met the guidelines for blood donation. Peripheral blood mononuclear cells (PBMCs) from healthy donors were prepared by density centrifugation on a Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Sweden). Monocytes were purified from PBMC by positive isolation using anti-CD14 conjugated magnetic microbeads (MACS CD14 isolation kit, Miltenyi Biotech, Bergisch Gladbach, Germany). Purity was checked by flow cytometer with anti-CD45-FITC and anti-CD14-PE antibodies, and was routinely > 95%.
Human monocyte-derived DCs were generated with GM-CSF (100 ng/ml) and IL-4 (20 ng/ml) for 6 days at 37°C in a 5% CO₂ atmosphere. Cultures were fed on Day 3 by adding fresh medium with cytokines. On Day 6, immature DCs (iDCs) were stimulated with LPS (500 ng/ml) to mature DCs (LPS-DC). In some experiments, TFF2 or IL-10 was added to investigate their inhibitory effect on DC maturation or function.

Monocytes, pan T cells, naïve CD4 T cells, and NK cells were obtained from peripheral blood mononuclear cells (PBMCs), isolated by Ficoll density gradient centrifugation (Lymphoprep, Serumwerk Bernburg) from buffy coats derived from healthy blood donors (Sanquin, the Netherlands). Monocytes were isolated with MACS CD14+ isolation kit (130-050-201, Miltenyi Biotec). After the positive CD14 isolation, the negative fraction containing the peripheral blood lymphocytes (PBLs) was used to isolate autologous pan T cells and NK cells. Pan T cells and NK cells were isolated using the MACS Pan T cell isolation kit (130-096-535, Miltenyi Biotec) and the NK cell isolation kit (130-092-657, Miltenyi Biotec) following manufacturer’s instructions. Naïve CD4 T cells were isolated from PBLs using the MACS Naïve CD4 T cell isolation kit (130-094-131, Miltenyi Biotec) following the manufacturer’s instructions. Pan T cells, NK cells, and naïve CD4 T cells were cryopreserved until use in freezing media, composed of 50% X-VIVO medium (Lonza), 40% FBS (HyClone) and 10% DMSO (WAK-Chemie).